The objective of this program is to identify and functionally characterize neurogenic genes that are required for CNS development. Given the high conservation in basic developmental mechanisms used by all metazoans, we have focused our efforts on the study of CNS development in the fruit fly (Drosophila melanogaster), where the genetic information required for these events is accessible. Using classical genetic, molecular biology and transgenic techniques, we have continued to study the function of genes expressed during neuroblast lineage differentiation. Thus far, our study of castor (cas), a novel Zinc finger gene required for proper CNS neuroblast development, has revealed that it encodes a nuclear located, sequence-specific DNA- binding protein whose expression is restricted to late forming CNS neuronal precursor sublineages. We hypothesize that the Cas protein functions as a transcription factor required for correct regulation of genes in these cells. Our recent studies have shown that cas controls cell fate decisions by regulating the expression of all known POU genes during CNS development. POU transcription factors establish neuronal identities in metazoans, yet little is known about the regulatory networks controlling their expression. Cas shares DNA-binding specificity with another pdm repressor, the gap-segmentation gene regulator Hunchback (Hb). Our studies reveal for the first time that all CNS ganglia contain sequentially layered neuroblast progeny subpopulations that can be distinguished by their expression of either Hb, Pdm-1 or Cas. By ensuring correct POU gene expression boundaries, hb and cas maintain temporal subdivisions in the cell- identity circuitry controlling CNS cellular diversity... Phenotypic analysis of loss of function mutations in hb, pdm or cas, by us and others, has shown each of these regulators carryout essential but distinct roles during NB lineage development. Due to the shared expression of these transcription factors, cells within a given expression domain may have overlapping repertoires of downstream target gene expression. To test this hypothesis, we are preparing cDNAs from isolated Hb, Pdm or Cas expressing neuroblasts. Using differential screening and in situ hybridization techniques we will search for genes whose expression is restricted to specific layers. By identifying additional target genes whose expression is restricted, we hope to not only expand our understanding of this regulatory network but also to connect these genetic circuits to genes those encoded proteins impart distinct neuronal phenotypes. - Developmental Neurobiology, Drosophila CNS Neuroblast Stem Cells